Cholinergic agents such as carbachol (CCh) are known to stimulate the secretion of HCO₃^- in the duodenum through an increase of intracellular Ca²⁺ via the activation of muscarinic acetylcholine receptors (mAChRs). In the present study, we investigated the roles of mAChRs subtypes in the cholinergic regulation of HCO₃^- secretion using mAChR M1~M5 knockout (KO) mice and demonstrated the importance of M4-receptor in the response to CCh. C57BL/6J mice of wild-type (WT) and M1~M5-KO were used. The duodenal mucosa, stripped of the muscular layers, was mounted on an Ussing chamber and bathed in saline gassed with 100% O2 on the mucosal side and HCO₃^--Ringer's solution gassed 95% O2/5% CO2 on the serosal side, and HCO₃^- secretion was measured at pH 7.0 using a pH-stat method. CCh (10-5~10-4 M) stimulated duodenal HCO₃^- secretion in a dose-dependent manner, and this action was totally inhibited by atropine given serosally 30 min before CCh. The HCO₃^- response to CCh was similarly observed in M2- and M5-KO mice, while significantly decreased in M1-, M3- and M4-KO mice. The decreased response in M4-KO mice, but not M1- or M3-KO mice, was reversed by the co-addition of CYN154806 the SST2 receptor antagonist. Somatostatin decreased the HCO₃^- response to CCh, in a CYN154806- inhibitable manner. Immunohistochemial study revealed the localization of M4 receptors in the duodenal D cells. The medium somatostatin level in the WT mouse duodenum was decreased in response to CCh. The duodenal HCO₃^- response to CCh is mainly mediated by the activation of M1 and M3 receptors and modified by M4 receptors but does not involve other mAChR subtypes. Somatostatin has an inhibitory effect on the HCO₃^- secretion through SST2 receptors. It is assumed that the activation of M4 receptors inhibits the release of somatostatin from D cells and results in enhancement of the HCO₃^- response by minimizing the negative influence of somatostatin.